1. Field of the Invention
This application is directed to a method of isolating a polypeptide in non-native form from cells in which it is made. More particularly, the invention relates to a method of isolating a polypeptide in non-native conformation from cells using a multiple-phase aqueous isolation technique.
2. Description of Related and Background Art
The use of recombinant DNA techniques to express DNA encoding heterologous protein has opened new possibilities to produce protein products in commercial quantities. By these methods, the gene encoding the product of interest is introduced into a host cell, e.g., bacteria, fungi, yeast, or mammalian cells, which can be grown in culture so that the gene will become expressed in the cell. Polypeptides so produced can be purified and used for a number of applications, including pharmaceutical and veterinarian uses and, in the case of enzymes, food industry or detergent uses.
Producing recombinant protein involves transforming or transfecting host cells with DNA encoding the desired exogenous protein and growing the cells and placing them under conditions favoring production of the recombinant protein. The prokaryote E. coli is favored as host because it can be made to produce recombinant proteins at high titers. Numerous U.S. patents on general bacterial production of recombinant-DNA-encoded proteins exist, including U.S. Pat. No. 4,565,785 on a recombinant DNA molecule comprising a bacterial gene for an extracellular or periplasmic carrier protein and non-bacterial gene; U.S. Pat. No. 4,673,641 on co-production of a foreign polypeptide with an aggregate-forming polypeptide; U.S. Pat. No. 4,738,921 on an expression vector with a trp promoter/operator and trp LE fusion with a polypeptide such as IGF-I; U.S. Pat. No. 4,795,706 on expression control sequences to include with a foreign protein; and U.S. Pat. No. 4,710,473 on specific circular DNA plasmids such as those encoding IGF-I.
Under some conditions, certain heterologous proteins expressed in large quantities from bacterial hosts are precipitated within the cells in dense aggregates, recognized as bright spots visible within the enclosure of the cells under a phase-contrast microscope. These aggregates of precipitated proteins are referred to as "refractile bodies," and constitute a significant portion of the total cell protein. Brems et al., Biochemistry, 24: 7662 (1985). On the other hand, the aggregates of protein may not be visible under the phase-contrast microscope, and the term "inclusion body" is often used to refer to the aggregates of protein whether visible or not under the phase-contrast microscope.
Recovery of the protein from these bodies has presented numerous problems, such as how to separate the protein encased within the cell from the cellular material and proteins harboring it, and how to recover the inclusion body protein in biologically active form. The recovered proteins are often predominantly biologically inactive because they are folded into a three-dimensional conformation different from that of active protein. For example, misfolded IGF-I with different disulfide bond pairs than found in native IGF-I has significantly reduced biological activity. Raschdorf et al., Biomedical and Environmental Mass Spectroscopy, 16:3-8 (1988). Misfolding occurs either in the cell during fermentation or during the isolation procedure. Methods for refolding the proteins into the correct, biologically active conformation are essential for obtaining functional proteins.
In addition to proper refolding, another challenge faced by biochemists and cell biologists is the development of efficient separation methods, both for soluble substances such as proteins and nucleic acids, and for suspended particles, such as cell organelles and whole cells. Upon fermentation of a prokaryotic broth, for example, many complex particles are generated when the cells are disintegrated. Procedures for separating proteins from these mixtures are complicated, providing particles different in size, form, and chemical composition. Also, the particles may aggregate, dissociate, or generally change their state with time and physical or chemical treatment. There is a great need for mild and efficient fractionation methods, particular for those applications where the level of purity of the product must be very high, e.g., at least 99 percent for pharmaceuticals.
Proteins are typically purified by one or more chromatographic methods such as affinity chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, and reverse-phase high-pressure liquid chromatography. Before a crude extract containing a protein of interest is applied to a chromatography column, the protein extract containing the desired product must be separated from solids such as cells and cell debris. This is because all components applied to the column must be able to pass through the gel matrix. Otherwise, the solid components would clog the gel bed and eventually stop the liquid flow completely. Thus, a purification method must be included in the process scheme to separate the product from solid components and usefully from viscous components, such as cells, cell debris, and nucleic acids, respectively.
The most commonly used methods for this purpose are centrifugal separation or microfiltration or both, depending on the product, host cell type, and localization of the product (extracellular, intracellular, bacterial periplasm, etc.). Since a number of different components are present in a mixture, different methods may be needed utilizing different properties of the particles. For example, centrifugation methods, which separate according to size and density of particles, may be complemented by methods in which other properties, such as surface properties, comprise the separation parameter. One of these methods is distribution in a liquid-liquid two-phase system. In such a method, the phase systems may be obtained by mixing water with different polymers, so that they are compatible with particles and macromolecules from biological material.
Aqueous two-phase partitioning was introduced in 1956-1958 with applications for both cell particles and proteins. Since then, it has been applied to a host of different materials, such as plant and animal cells, microorganisms, viruses, chloroplasts, mitochondria, membrane vesicles, proteins, and nucleic acids.
The basis for separation by a two-phase system is selective distribution of substances between the phases. For a soluble substance, distribution occurs mainly between the two bulk phases, and the partitioning is characterized by the partition coefficient, which is defined as the concentration of partitioned substance in the top phase, divided by the concentration of the partitioned substance in the bottom phase. Ideally, the partition coefficient is independent of total concentration and the volume ratio of the phases. It is mainly a function of the properties of the two phases, the partitioned substance, and the temperature.
The two-phase systems may be produced by mixing two phase-incompatible polymer solutions, by mixing a polymer solution and a salt solution, or by mixing a salt solution and a slightly apolar solvent. These types of systems, along with aqueous two-phase partitioning methods for separating macromolecules such as proteins and nucleic acids, cell particles, and intact cells are described, for example, in Albertsson, Partition of Cell Particles and Macromolecules, 3rd edition (John Wiley & Sons: New York, 1986); Walter et al., Partitioning in Aqueous Two-Phase Systems: Theory, Methods, Uses, and Applications to Biotechnology, (Academic Press: London, 1985); and Kula, "Extraction processes--application to enzyme purification (conference paper)," 8th Int. Biotechnol. Symp. (pt. 1, 612-622), 1988.
Several low-cost two-phase systems are known that can handle protein separations on a large scale. These systems use polyethylene glycol (PEG) as the upper phase-forming polymer and crude dextran (e.g., Kroner et al., Biotechnology Bioengineering, 24:1015-1045 [1982]), a concentrated salt solution (e.g., Kula et al., Adv. Biochem. Bioeng., 24: 73-118 [1982]), or hydroxypropyl starch (Tjerneld et al., Biotechnology Bioengineering, 3.0:809-816 [1987]) as the lower phase-forming polymer.
Purification of interferon has been achieved by selective distribution of crude interferon solutions in aqueous PEG-dextran systems or PEG-salt systems using various PEG derivatives. German Patent DE 2,943,016.
Two-phase aqueous polymer systems are extensively discussed in the literature. See, e.g., Baskir et al., Macromolecules, 20: 1300-1311 (1987); Birkenmeier et al., J. Chromatogr., 360:193-201 (1986); Birkenmeier and Kopperschlaeger, J. Biotechnol., 21:93-108 (1991); Blomquist and Albertsson, J. Chromatogr., 73: 125-133 (1972); Blomquist et al., Acta Chem. Scand., 29: 838-842 (1975); Erlanson-Albertsson, Biochim. Biophys. Acta, 617: 371-382 (1980); Foster and Herr, Biol. Reprod., 46: 981-990 (1992); Glossmann and Gips, Naunyn. Schmiedebergs Arch. Pharmacol., 282: 439-444 (1974); Hattori and Iwasaki, J. Biochem. (Tokyo), 88: 725-736 (1980); Haynes et al., AICHE Journal-American Institute of Chemical Engineers, 37: 1401-1409 (1991); Johansson et al., J. Chromatogr., 331: 11-21 (1985); Johansson et al., J. Chromatogr., 331: 11-21 (1985); Kessel and McElhinney, Mol. Pharmacol., 14: 1121-1129 (1978); Kowalczyk and Bandurski, Biochemical Journal, 279: 509-514 (1991); Ku et al., Biotechnol. Bioeng., 33: 1081-1088 (1989); Kuboi et al., Kagaku Kogaku Ronbunshu, 16: 1053-1059 (1990); Kuboi et al., Kagaku Kogaku Ronbunshu, 16: 755-762 (1990); Kuboi et al., Kagaku Kogaku Ronbunshu, 17: 67-74 (1991); Kuboi et al., Kagaku Kogaku Ronbunshu, 16: 772-779 (1990); Lemoine et al., Physiol. Plant, 82: 377-384 (1991); Lillehoj and Malik, Adv. Biochem. Eng. Biotechnol., 40: 19-71 (1989); Lundberg et al., Biochemistry, 31: 5665-5671 (1992); Marciani and Bader, Biochim. Biophys. Acta, 401: 386-398 (1975); Mattiasson and Kaul, "Use of aqueous two-phase systems for recovery and purification in biotechnology" (conference paper), 314, Separ. Recovery Purif.: Math. Model., 78-92 (1986); Mendieta and Johansson, Anal. Biochem., 200: 280-285 (1992); Nifant'eva et al., Zh. Anal. Khim., 44:1368-1373 (1989); O'Brien et al., Blood, 80: 277-285 (1992); Ohlsson et al., Nucl. Acids Res., 5: 583-590 (1978); Owusu and Cowan, Enzyme Microb. Technol., 11: 568-574 (1989); Pruul et al., J. Med. Microbiol., 32: 93-100 (1990); Sandstrom et al., Plant Physiol. (Bethesda), 85: 693-698 (1987); Sasakawa and Walter, Biochim. Biophys. Acta, 244:461-465 (1971); Wang et al., J. Chem. Engineering of Japan, 25: 134-139 (1992); Widell and Sundqvist, Physiol. Plant, 61: 27-34 (1984); Zaslavskii et al., J. Chrom., 439: 267-281 (1988); Zaslavskii et al., J. Chem. Soc., Faraday Trans., 87:141-145 (1991); U.S. Pat. No. 4,879,234 issued Nov. 7, 1989 (equivalent to EP 210,532); DD (German) 298,424 published Feb. 20, 1992; WO 92/07868 published May 14, 1992; and U.S. Pat. No. 5,093,254. See also Hejnaes et al., Protein Engineering, 5: 797-806 (1992).
An aqueous two-phase extraction/isolation system is described by DD Pat. No. 288,837. In this process for selective enrichment of recombinant proteins, a protein-containing homogenate is suspended in an aqueous two-phase system consisting of PEG and polyvinyl alcohol as phase-incompatible polymers. Phase separation is then performed whereby the protein is concentrated in the top phase while most of the biomass is concentrated in the bottom phase. However, this patent does not address how to partition non-native proteins.
Cole, Biotechniques, 11: 18-24 (1991) adds chaotropes and detergents to a two-phase aqueous system to inactivate nucleases that might degrade the DNA being isolated. Cole, Frontiers Bioprocess II, 340-351 (1992) and Grunfeld et al., Appl. Biochem. Biotechnol., 33: 117-138 (1992) use a two-phase system for reactivation of t-PA or for purification of t-PA from its reactivation mixture. Johansson and Kopperschlaeger, J. Chrom., 388: 295-305 (1987) mention urea as reducing the affinity partitioning effect for alkaline phosphatase. Mak et al., Biochemistry, 15: 5754-5761 (1976) purifies RNA using an aqueous polymer two-phase system. Moudgil et al., J. Biol. Chem., 262: 5180-5187 (1987) uses urea or heat to alter the partition coefficient for a receptor. Niwa et al., Nippon Suisan Gakkaishi-Bulletin of the Jap. Soc. of Scientific Fisheries, 55: 143-146 (1989); Tanaka et al., J. Chem. Eng. Jpn., 24: 661-664 (1991); and WO 91/02089 published Feb. 21, 1991 report on extraction of nucleic acids. See also U.S. Pat. No. 4,843,155.
The main benefits of the partitioning technique are the method is efficient, easy to scale up, rapid when used with continuous centrifugal separators, relatively low in cost, and high in water content to maximize biocompatibility. Although considerable savings can be made by their use, there are currently relatively few industrial applications of aqueous two-phase systems to purify proteins.
When a protein is to be isolated from a crude extract by two-phase partitioning, recovery is enhanced by having a maximum distribution of the protein between the phases. A large or small partition coefficient relative to that for the rest of the cell protein provides a means for purifying the product. It is further possible to isolate the product using an extreme phase volume ratio, with a volume reduction as the result, and still retain a high yield. When the partition coefficient is high, as is the case for the intracellular enzyme .beta.-galactosidase, the aqueous two-phase partitioning will provide for a purification and concentration of the product, in addition to removing cell particles and nucleic acids in one step. Thus, it is possible to collect a product in a PEG-rich top phase, and at the same time displace cell particles and nucleic acids into the salt-rich bottom phase.
The search for extreme partition coefficients, which provide the possibility for achieving a concentrated product with a high yield, has led to use of a number of second-generation aqueous two-phase systems. One example is affinity partitioning, where PEG is covalently coupled to affinity groups and the resulting conjugate is included as a polymer component to enhance partitioning to the PEG-rich phase. These, and similar approaches, can make the aqueous two-phase extraction/isolation very selective for essentially any product. However, the high cost of the modified PEG, problems in finding a suitable affinity group, and the necessity to recycle the modified PEG for economical reasons, make this concept unattractive for large-scale applications. Another concept has been to fuse the product of interest to a protein that has a large partition coefficient, or to a peptide sequence containing tryptophan residues, as described by WO 92/7868.
There is a need in the art for a method for directly isolating recombinant polypeptides from culture in situ in the fermentation tank, without requiring that the protein be renatured and without requiring costly ingredients such as derivatized polymers or fusions of product with a peptide or other affinity agent.
Therefore, it is an object of the present invention to provide a procedure for isolating non-native polypeptides from a broth in which they exist with other species.
It is another object to provide an efficient extraction of recombinant proteins from homogenates of fermentation broth.
It is a specific object to provide a multiple-phase aqueous isolation composition for non-native IGF-I from a tank containing the recombinant protein in the form of inclusion bodies.
These and other objects will be apparent to one of ordinary skill in the art.